The invention relates to methods of genetically transforming plant plastids, and more specifically to genetically transforming the plastid genomes of Solanaceous plant species.
The plastids of higher plants are an attractive target for genetic engineering. Plant plastids (chloroplasts, amyloplasts, elaioplasts, etioplasts, chromoplasts, etc.) are the major biosynthetic centers that, in addition to photosynthesis, are responsible for production of industrially important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments. Plastids are derived from a common precursor known as a proplastid and thus the plastids present in a given plant species all have the same genetic content. In general, plant cells contain 500-10,000 copies of a small 120-160 kilobase circular genome, each molecule of which has a large (approximately 25 kb) inverted repeat. Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest which potentially can result in very high levels of foreign gene expression. In addition, plastids of most plants are maternally inherited. Consequently, unlike heterologous genes expressed in the nucleus, heterologous genes expressed in plastids are not pollen disseminated, therefore, a trait introduced into a plant plastid will not be transmitted to wild-type relatives.
Unfortunately, successful chloroplast transformation techniques described thusfar have been limited to tobacco (U.S. Pat. No.5,451,513; Svab et. al. (1990), Proc. Natl. Acad. Sci. USA 87:8526-8530 and Svab et al. (1993), Proc. Natl. Acad. Sci. USA 90:913-197). For practical applications of genetic engineering techniques to crop plant plastids, chloroplast transformation techniques for a wide variety of crop plants are needed in the art.
The genus Solanaceae includes many agriculturally important plants, and includes some 95 genera. Solanaceous lid crop plants include potato, tomato, eggplant, and other lesser known edible fruits from Physalis (cape gooseberry, strawberry tomato, jamberberry, sugar cherry, chinese lantern, etc), tamarillo, and Capsicum (sweet and chili peppers). The genus also includes many cultivated ornamentals, for example, Petunia, Lycium, Solanum, and Solandra. Other important crops from the genus Solanaceae include tobacco (Nicotiana) and other poisonous alkaloid producing plants such as Hyoscyamus and Datura.
Plastids of higher plants present an attractive target for genetic engineering. As mentioned above, plastids of higher plants are maternally inherited. This offers an advantage for genetic engineering of plants for tolerance or resistance to natural or chemical conditions, such as herbicide tolerance, as these traits will not be transmitted to wild-type relatives.
The production of chloroplast transformation methods applicable to crop species other than tobacco is needed in the art. Such methods provide for a novel means of genetic engineering via plastid transformation to an attractive alternative to nuclear expression of agronomically as well as qualitatively important traits via genetic engineering of plant plastids.
Relevant Literature
Stable transformation of plastids has been reported in the green algae Chlamydomonas (Boynton et al. (1988) Science 240:1534-1538) and most recently in higher plants (Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917). These methods rely on particle gun delivery of DNA containing a selectable marker and targeting to the plastid genome by homologous recombination.
The complete DNA sequences of the plastid genomes from liverwort (Ohyama et al. (1986) Nature 322:572-574), rice (Hiratsuka et al. (1989) Mol. Gen. Genet. 217:185-194), and tobacco (Shinozaki et al. (1986) EMBO J. 5:2043-2049) have been reported.
The present invention provides methods for the transformation and regeneration of plants containing plant cells, the plastids of which have been stably transformed by a foreign DNA of interest. The method generally comprises transforming a Solanaceous plant cell plastid with a DNA construct; selecting for cells which contain the DNA construct; and obtaining a mature multicellular plant, the cells of which contain the DNA construct in the plant cell plastid.
The instant invention also provides methods for transforming the plastids of Solanaceous plant cells with a DNA construct generally comprising, in the 5xe2x80x2 to 3xe2x80x2 direction of transcription, a promoter region functional in a plant cell plastid, a DNA sequence of interest, and a transcription termination region functional in a plant cell plastid.
Furthermore, the present invention also provides the multicellular solanaceous plant obtained by the methods described herein.
The invention also provides a multicellular solanaceous plant, the plastids of which have been transformed with a DNA construct of interest.
The invention also provides a method for obtaining a plant cell, of which the plastid has been stably transformed with a DNA construct, comprising in the 5xe2x80x2 to 3xe2x80x2 direction of transcription, a promoter functional in a plant cell plastid, a DNA sequence encoding a green fluorescent protein (herein referred to as GFP), and a transcriptional termination region functional in a plant cell plastid.
The invention also provides for the multicellular plant, the plastids of which have been transformed with a DNA construct comprising in the 5xe2x80x2 to 3xe2x80x2 direction of transcription, a promoter functional in a plant cell plastid, a DNA sequence encoding a green fluorescent protein (herein referred to as GFP), and a transcriptional termination region functional in a plant cell plastid.